Methods for detecting DNA originating from different individuals

ABSTRACT

In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.

BACKGROUND OF THE INVENTION

The presence of DNA originating from different individuals in bodilyfluids is a well-known biological phenomenon in many clinical andbiological scenarios. For example, following bone marrowtransplantation, the hemopoietic system of the transplantation recipientwill consist of varying proportions of donor's and recipient's cells.The ascertainment of the amount of donor's or recipient's cells has beenperformed by the detection of genetic differences between the donor andrecipient, including gender (Mangioni et al., Bone Marrow Transplant20:969-73 (1997)) and DNA polymorphisms (Roux et al., Blood 79:2775-83(1992)). The corollary of this approach is that if the analysed regiondoes not bear a genetic difference between the donor and recipient, thenanalysis by the current approach will not be possible.

In another example, during pregnancy, detection of fetal DNA in maternalplasma and serum has been previously demonstrated (Lo et al., Lancet350:9076: 485-7(1997)). This technology has demonstrated that fetal DNAisolated from maternal plasma and serum can be used for non-invasiveprenatal diagnosis (Lo et al., N Eng J Med, 339(24):1734-8 (1998); Faaset al., Lancet 352(9135):1196 (1998); Amicucci et al., Clin Chem46(2):301 (2000); Chen et al., Prenat Diagn 204:355-7 (2000); Saito etal., Lancet 356:1170 (2000)). The clinical application of thisphenomenon has been helped by the relatively high absolute and relativeconcentrations of such circulating fetal DNA in maternal plasma andserum (Lo et al., Am J Hum Genet 62:768-775 (1998)). Using thisapproach, noninvasive prenatal detection of a number of conditions hasbeen achieved, including fetal rhesus D status (Lo et al., New Eng J Med339:1734-1738 (1998)), myotonic dystrophy (Amicucci et al., Clin Chem46:301-302 (2000)), achondroplasia (Saito et al., Lancet 356:1170(2000)) and certain chromosomal translocations (Chen et al., Prenat Diag20:335-357 (2000); Chen et al., Clin Chem 47:937-939 (2001)). All ofthese current approaches have utilized the detection of DNA sequencesinherited from the father and which are genetically distinguishable fromthose of the mother (Bianchi, Am J Hum Genet 62(4): 763 (1998).Specifically, the detection of DNA that the fetus has inherited from themother in maternal plasma or serum has been thought to be impossible.Similar limitations have also been described for the detection of fetalnucleated cells isolated from the cellular fraction of maternal blood(Lo et al., Ann NY Acad Sci, 731:204 (1994).

Others have detected aberrantly methylated DNA from cancer patients.This has been reported for patients with a variety of cancers, includinglung (Esteller, et al., Cancer Res 59(1):67 (1999)) and liver cancer(Wong et al., Cancer Res 59(1):71 (1999)).

Recently, much interest has been focused on the biology of epigeneticphenomena, namely processes which alter the phenotype but which are notassociated with changes in DNA sequence (Wolffe, Science 286:481-486(1999)). One of the best characterised epigenetic processes is DNAmethylation (Wolffe et al., Curr Biol. 10:R463-R465 (1999)). A methodfor discriminating DNA species originating from different individuals inbiological fluids using epigenetic, rather than genetic differencesbetween the DNA species would be highly valuable. For example, theepigenetic detection of fetal DNA in a maternal sample would provide asignificant advancement enabling additional screening and diagnosticmethods.

SUMMARY OF THE INVENTION

In a first aspect, the present invention features methods fordifferentiating DNA species originating from different individuals in abiological sample. In preferred embodiments the methods of the presentinvention are used to differentiate or detect fetal DNA in a maternalsample or to differentiate DNA of an organ donor from DNA of an organrecipient.

Those of skill in the art will appreciate that the biological sampleobtained from an individual may be taken from any fluid or cell sample,however, in preferred embodiments the bodily fluid is plasma or serum.In preferred embodiments, the DNA species are differentiated byobserving epigenetic differences in the DNA species such as differencesin DNA methylation. For instance, in situations where one DNA speciescomes from a male, and one DNA species comes from a female, theepigenetic marker may be the inactivated X chromosome of the femaleindividual. In such embodiments, methylated DNA sequences on theinactivated X chromosome may be used to detect DNA originating from thefemale individual. In some embodiments, the epigenetic differences maybe analyzed inside cells. Further, in some embodiments, the epigeneticdifferences may be analyzed using in-situ methylation-specificpolymerase chain reaction. Additionally, the epigenetic differences maybe used to sort or isolate cells from the respective individuals or topurify DNA from the respective individuals. The methods according to thepresent invention may be performed with or without measuring theconcentrations of DNA species, however, in preferred embodiments, theconcentrations of DNA species with the respective epigenetic differencesare measured. Such measuring of concentrations involves measuring therespective DNA methylation differences in embodiments wherein DNAmethylation differences is the epigenetic marker. In especiallypreferred embodiments, sodium bisulfite is added to the biologicalsample or to the DNA species directly to detect the DNA methylationdifferences. However, in other embodiments a methylation-specificpolymerase chain reaction, as is well known to those skilled in the art,may be used to detect the DNA methylation differences. In yet otherembodiments, DNA sequencing or primer extension may be used to detectthe methylation differences.

In a second aspect, the present invention features methods of detectingabnormalities in a fetus by detecting fetal DNA in a biological sampleobtained from a mother. The methods according to the present inventionprovide for detecting fetal DNA in a maternal sample by differentiatingthe fetal DNA from the maternal DNA based upon epigenetic markers suchas differences in DNA methylation. Employing such methods, fetal DNAthat is predictive of a genetic anomaly or genetically based disease maybe identified thereby providing methods for prenatal diagnosis. Thesemethods are applicable to any and all pregnancy-associated conditionsfor which methylation changes associated with a disease state isidentified. Exemplary diseases that may be diagnosed include, forexample, preeclampsia, a chromosomal aneuploidy, including but notlimited to trisomy 21, Prader-Willi Syndrome, and Angelman Syndrome.

As with the broader differentiating methods of the first aspect of theinvention, the biological sample obtained from the mother is preferablyplasma or serum. The differentiation between maternal and fetal DNA maybe performed with or without quantifying the concentration of fetal DNAin maternal plasma or serum. In embodiments wherein the fetal DNA isquantified, the measured concentration may be used to predict, monitoror diagnose or prognosticate a pregnancy-associated disorder. Inpreferred embodiments, the particular fetus-derived epigenetic mark isassociated with a fetal disorder, and in some embodiments an epigeneticcharacteristic in fetal cells in the placenta is used as afetus-specific marker in maternal plasma or serum.

In a third aspect, the present invention features methods fordifferentiating DNA species originating from an organ donor from thoseof an organ recipient. As with the broader differentiating methods ofthe first aspect of the invention, the biological sample obtained ispreferably plasma or serum. The differentiation between DNA from theorgan donor and organ recipient or potential organ donor and potentialorgan recipient may be performed with or without quantifying theconcentration of DNA in the biological sample. This embodiment isparticularly useful in instances when the transplantation is a bonemarrow transplantation. Such measurements may be used to predict theclinical progress of the transplantation recipient especially as regardsorgan rejection.

In a fourth aspect, the present invention features kits fordifferentiating DNA species originating from different individuals in abiological sample. Such kits are useful, for instance, fordifferentiating or detecting the presence of fetal DNA in a maternalbiological sample or for differentiating DNA from an organ donor orpotential organ donor from that of an organ recipient or potential organrecipient. The kits according to the present invention comprise one ormore reagents for ascertaining the methylation status of the maternalDNA such as sodium bisulfite and one or more reagents for detecting thepresence of DNA such as a gel. Additionally, such kits may include oneor more reagents for amplifying the amount of DNA present in the samplesuch as one or more reagents for performing polymerase chain reactionamplification. Such reagents are well known to those of skill in theart. Further, such kits may include one or more apparatuses forobtaining a maternal DNA sample. Such apparatuses are well known tothose skilled in the art. In particular the kits according to thepresent invention may be used for diagnosing a disease caused all or inpart by a genetic anomaly such as a mutation, substitution or deletionin all or part of a DNA sequence present in a fetus. Exemplary diseasesthat may be diagnosed include, for example, preeclampsia, a chromosomalaneuploidy, including but not limited to trisomy 21, Prader-WilliSyndrome and Angelman Syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the results of an assay detecting methylated andunmethylated DNA sequences of the androgen receptor gene. In total, 6male and 11 female healthy subjects were recruited. Of all male controlsubjects, only the unmethylated androgen receptor gene was detected inthese samples as expected (FIG. 1A). By contrast, both unmethylated andmethylated androgen receptor gene DNA sequences were observed in femalecontrol subjects (FIG. 1A). The detection rates of methylated andunmethylated androgen receptor genes in these female subjects were 100%and 82%, respectively. When DNA samples were omitted from the assay, nopositive signal was observed (FIG. 1A). Interestingly, positive signalsfor both methylated and unmethylated DNA sequences were observed in allmale bone marrow transplantation recipients with female donors,indicating cells from female donor exist in the blood circulation ofmale recipients.

FIG. 2 provides a schematic representation of the differentiallymethylated region (DMR) of the human IGF2-H19 region. The two 450-bprepeat (A1 and A2) and seven 400-bp repeat (B1-B7) units are shown. Thepotential methylation sites on the upper strand DNA of the studiedregion are represented by open circles. The studied single nucleotidepolymorphism (SNP) site (A/G) is indicated by an open box. Open arrowsrepresent the location of the forward (for) and reverse (rev) primers inPCR reactions specific for the methylated (M) and unmethylated (U)alleles, respectively. Sequences of these MSP primers are shown.Sequence differences between bisulfite-treated DNA and untreated DNA arehighlighted in bold italics and sequence differences between methylated(paternally-inherited) and unmethylated (maternally-inherited) DNA areunderlined in bold.

FIG. 3 demonstrates detection of methylated (paternally-inherited) fetalDNA in 3^(rd) trimester (a) and 2^(nd) trimester (b) maternal plasma.DNA sequence of methylated alleles in maternal buffy coat (panel 1),fetal buffy coat or amniotic fluid (panel 2), prenatal maternal plasma(panel 3) and postnatal maternal plasma (panel 4) samples are shown. Thepresence of methylated fetal DNA in the prenatal maternal plasma sampleis indicated by *. The polymorphic (SNP) site is shown in red letters.

FIG. 4 demonstrates detection of unmethylated (maternally-inherited)fetal DNA in maternal plasma. (a) Unmethylated DNA sequences weredetected in maternal buffy coat (panel 1) and a third trimester maternalsample (panel 2) using direct sequencing. The presence of unmethylatedfetal DNA in maternal plasma is indicated by *. (b) Unmethylated fetalDNA (arrow) was detected in two third trimester maternal plasma samplesusing the primer extension assay. (c) Unmethylated fetal DNA (arrow) wasdetected in a second trimester maternal plasma sample using the primerextension assay. Products from control reactions containing primer only,unmethylated G allele or unmethylated A allele are shown. The sizes (nt)of the reaction products are shown at the bottom. ●, unused primer; □detected allele.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In a first aspect, the present invention features methods fordifferentiating DNA species originating from different individuals in abiological sample. In preferred embodiments the methods of the presentinvention are used to differentiate or detect fetal DNA in a maternalsample or to differentiate DNA of an organ donor from DNA of an organrecipient.

Those of skill in the art will appreciate that the biological sampleobtained from an individual may be taken from any fluid or cell sample,however, in preferred embodiments the bodily fluid is plasma or serum.In preferred embodiments, the DNA species are differentiated byobserving epigenetic differences in the DNA species such as differencesin DNA methylation. For instance, in situations where one DNA speciescomes from a male, and one DNA species comes from a female, theepigenetic marker may be the inactivated X chromosome of the femaleindividual. In such embodiments, methylated DNA sequences on theinactivated X chromosome may be used to detect DNA originating from thefemale individual. In some embodiments, the epigenetic differences maybe analyzed inside cells. Further, in some embodiments, the epigeneticdifferences may be analyzed using in-situ methylation-specificpolymerase chain reaction. Additionally, the epigenetic differences maybe used to sort or isolate cells from the respective individuals or topurify DNA from the respective individuals. The methods according to thepresent invention may be performed with or without measuring theconcentrations of DNA species, however, in preferred embodiments, theconcentrations of DNA species with the respective epigenetic differencesare measured. Such measuring of concentrations involves measuring therespective DNA methylation differences in embodiments wherein DNAmethylation differences is the epigenetic marker. In especiallypreferred embodiments, sodium bisulfite is added to the biologicalsample or to the DNA species directly to detect the DNA methylationdifferences. However, in other embodiments a methylation-specificpolymerase chain reaction, as is well known to those skilled in the art,may be used to detect the DNA methylation differences. In yet otherembodiments, DNA sequencing or primer extension may be used to detectthe methylation differences.

As used herein, the term “biological sample” is intended to encompassany fluid or cellular sample or mixture thereof obtained from a livingorganism. Specifically, the term includes tissue biopsy, serum, plasmaor amniotic fluid samples.

As used herein, the term “epigenetic difference” is intended toencompass any molecular or structural difference other than the primarynucleotide sequence. For instance, this may include differences inmethylation.

As used herein, the term “DNA” is intended to encompass any sequence ofmore than one nucleotide such as polynucleotides, gene fragments andcomplete gene sequences.

As used herein, the term “methylation-specific PCR” is used to describea method in which DNA is treated with sodium bisulfite and thensubjected to PCR amplification. This technique is based on the principlethat treating DNA with bisulfite results in conversion of unmethylatedcytosine residues into uracil. Methylated cytosine residues, on theother hand, remain unchanged. Thus, the DNA sequences of methylated andunmethylated genomic regions following bisulfite conversion aredifferent and distinguishable by sequence-specific PCR primers.

The present invention utilizes the phenomenon of genomic imprinting toovercome the limitations of the prior art. In genomic imprinting, DNAsequences are modified biochemically, without alteration in DNAsequence. If this process results in differential modification of thefetal and maternal DNA, then this difference can be exploited for thediscrimination of fetal from maternal DNA in maternal plasma and serum.This phenomenon can also be used for the discrimination of fetal cellsfrom maternal cells in the cellular fraction of maternal blood. Inaddition, this principle can also be used to detect maternal cells orDNA that has entered into the body of the fetus/baby (Lo, et al., Blood88(11):4390-5(1996); Lo, et al., Cin Chem, 46(9):1301-9 (2000); Maloneyet al., J Clin Invest 104(1):41-7 (1999). This phenomenon can also beused in many other clinical scenarios wherein cells or DNA sequences arefound to be present inside the body of an individual, such as followingbone marrow transplantation (Lo et al., Br J Haematol 89(3):645-9(1995)) or solid organ transplantation (Starzl et al., Curr Opin NephrolHypertens 6(3):292-8 (1997); Lo et al., Lancet 351(9112):1329-30 (1998);Zhang, Clin Chem 45(10):1741-6 (1999)).

The present invention allows development of a gender-independent andpolymorphism-independent marker for fetal DNA in maternal plasma/serum.To develop a gender-independent and polymorphism-independent fetalmarker, one can use DNA sequences which are preferentially andspecifically methylated in the trophoblasts (Ohgane et al., Dev Genet,22(2): 132-40 (1998)). This overcomes the current limitation which canonly easily detect the presence of DNA from a male fetus in theplasma/serum of the mother (by using the Y-chromosome as the target)(Lo, et al., Am J Hum Genet, 62(4):768 (1998). It provides detectionmethods separate from relying on sequence differences in fetal andmaternal DNA to make such a distinction (Tang et al., Clin Chem45(11):2033-5 (1999); Pertl et al., Hum Genet 106:45-49 (2000)).

The development of molecular detection methods such as the PCR hasprovided many powerful tools for the monitoring of chimerism followingbone marrow transplantation (BMT). One of the most widely used PCR-basedtests for the detection of post-BMT chimerism in sex-mismatched cases isPCR for sequences on the Y chromosome (Lo et al., Br J Haematol 89:645-9 (1995). The limitation of this strategy is that it can only beused in cases wherein the donor is male and the recipient is female. Thepresent invention provides a system that can be applied to situationswhen the donor is female and the recipient is male. The fact that thephenomenon of Lyonization only exists in females, can be exploited todevelop a female-specific marker. In this phenomenon, one of the two Xchromosomes in a female individual is inactivated at random, withmethylation occurring in a number of the inactivated genes. Thistherefore allows an assay for detecting female DNA in an excess of maleDNA and which can be applied to BMT with female donors and malerecipients.

In a second aspect, the present invention features methods of detectingabnormalities in a fetus by detecting fetal DNA in a biological sampleobtained from a mother. The methods according to the present inventionprovide for detecting fetal DNA in a maternal sample by differentiatingthe fetal DNA from the maternal DNA based upon epigenetic markers suchas differences in DNA methylation. Employing such methods, fetal DNAthat is predictive of an anomaly or a disease may be identified therebyproviding methods for prenatal diagnosis. These methods are applicableto any and all pregnancy-associated conditions for which methylationchanges associated with a disease state is identified. Exemplarydiseases that may be diagnosed include, for example, preeclampsia, achromosomal aneuploidy, including but not limited to trisomy 21,Prader-Willi Syndrome, and Angelman Syndrome.

As with the broader differentiating methods of the first aspect of theinvention, the biological sample obtained from the mother is preferablyplasma or serum. The differentiation between maternal and fetal DNA maybe performed with or without quantifying the concentration of fetal DNAin maternal plasma or serum. In embodiments, wherein the fetal DNA isquantified, the measured concentration may be used to predict, monitoror diagnose a pregnancy-associated disorder. In preferred embodiments,the particular fetus-derived epigenetic mark is associated with a fetaldisorder, and in some embodiments an epigenetic characterisitic in fetalcells in the placenta is used as a fetus-specific marker in maternalplasma or serum.

The present invention utilizes differentially methylated fetal DNAsequences, which do not need to be distinguishable in terms of DNAsequence from maternal DNA, as markers for non-invasive prenataldiagnosis. This novel approach can convert fetus-mother pairs who arenot informative in the conventional approach, to being informative forprenatal diagnosis. Thus, present invention provides a platform on whicha new generation of non-invasive prenatal tests can be built.

The methods of the present invention are based on the detection ofdifferently methylated DNA of fetal origin in the plasma or serum ofpregnant women. Differentially methylated DNA sequences, which maycontain single nucleotide polymorphism, are preferably detected bymethylation-specific polymerase chain reaction (PCR); but in principleany detection method for differentially methylated DNA can be used. Thisapproach allows the use of conventional uninformative fetal DNA markersfor prenatal diagnosis.

The present invention allows detecting or predicting the presence of anydisorders of the fetus or the mother which are associated with a changein methylation status of a DNA sequence. Examples include imprintingdisorders such as Prader-Willi syndrome (Kubota et al., Nat Genet16(1):16-7 (1997). The present invention provides a new type of test forpreeclampsia which has been suggested to be an imprinting disorder(Graves, Reprod Fertil Dev 10(1):23-9 (1998). The present inventionfurther provides a new type of test for chromosomal aneuploidies,including Down syndrome (trisomy 21), which may be associated withmethylation changes (Yu et al., Proc Natl Acad Sci USA 94(13):6862-7(1997).

The present invention features using DNA methylation differences betweenthe mother and fetus thereby overcoming the limitations of the prior artin the detection of fetal DNA in maternal plasma.

In a third aspect, the present invention features methods fordifferentiating DNA species originating from an organ donor from thoseof an organ recipient. As with the broader differentiating methods ofthe first aspect of the invention, the biological sample obtained ispreferably plasma or serum. The differentiation between DNA from theorgan donor and organ recipient or potential organ donor and potentialorgan recipient may be performed with or without quantifying theconcentration of DNA in the biological sample. This embodiment isparticularly useful in instances when the transplantation is a bonemarrow transplantation. Such measurements may be used to predict theclinical progress of the transplantation recipient especially as appliedto organ rejection.

In a fourth aspect, the present invention features kits fordifferentiating DNA species originating from different individuals in abiological sample. Such kits are useful, for instance, fordifferentiating or detecting the presence of fetal DNA in a maternalbiological sample or for differentiating DNA from an organ donor orpotential organ donor from that of an organ recipient or potential organrecipient. The kits according to the present invention comprise one ormore reagents for ascertaining the methylation status of the maternalDNA such as sodium bisulfite and one or more reagents for detecting thepresence of DNA such as a gel. Additionally, such kits may include oneor more reagents for amplifying the amount of DNA present in the samplesuch as one or more reagents for performing polymerase chain reactionamplification. Such reagents are well known to those of skill in theart. Further, such kits may include one or more apparatuses forobtaining a maternal DNA sample. Such apparatuses are well known tothose skilled in the art. In particular the kits according to thepresent invention may be used for diagnosing a disease caused all or inpart by a genetic anomaly such as a mutation, substitution or deletionor duplication in all or part of a DNA sequence present in a fetus.Exemplary diseases that may be diagnosed include, for example,preeclampsia, a chromosomal aneuploidy, including but not limited totrisomy 21, Prader-Willi Syndrome and Angelman Syndrome.

EXAMPLE 1

Detection of Post-Bone Marrow Transplantation Chimerism Using a NovelEpigenetic Approach

Materials and Methods

Subjects and Samples

Four male marrow transplantation recipients, who received bone marrowfrom female donors, and 17 normal healthy subjects were recruited inthis study. Buffy coat (BC) from all recruited EDTA-blood samples wereharvested and stored at −20° C. as described (Lo et al., Am J Hum Genet62:768-75 (1998).

DNA Isolation

DNA was extracted from the BC using a Nucleon DNA Extraction Kit(Scotlabs) according to manufacturer's recommendations.

Bisulfite Conversion

Bisulfite modification of DNA samples was performed using a CpGenome DNAModification Kit (Intergen) as instructed by the manufacturer. Withbisulfite conversion, unmethylated cytosine residues are converted touracil while methylated cytosine residues remain unchanged (Herman etal., Proc Natl Acad Sci USA 93:9821-6 (1996). The sequence differencebetween methylated and unmethylated DNA following bisulfite conversionis then distinguished using different PCR primers. 1 μg of BC DNA wasused in a bisulfite conversion reaction.

Methylation-Specific PCR (MSP)

MSP assays were modified from the protocol as described by Herman et al,supra. The primers M-for (5′-GCGAGCGTAGTATTTTTCGGC-3′) and M-rev(5′-AACCAAATAACCTATAAAACCTCTACG-3′) were designed for the methylatedsequence, while the primers U-for (5′-GTTGTGAGTGTAGTATTTTTTGGT-3′) andU-rev (5′-CAAATAACCTATAAAACCTCTACA-3′) were designed for theunmethylated sequence. Five μl bisulfite-treated DNA was added to a 50μl PCR reaction containing 5 μl 10× TaqMan buffer A (PE AppliedBiosystems), 2 mM MgCl₂, 10 pmol dNTPs, 20 pmol each of thecorresponding MSP primers and 1.25 U AmpliTaq Gold DNA polymerase (PEApplied Biosystems). Reaction mixtures were thermal cycled (methylatedallele: 95° C. for 45 sec, 58° C. for 30 sec, 72° C. for 20 sec;unmethylated allele: 95° C. for 45 sec, 50° C. for 30 sec, 72° C. for 20sec) for 45 cycles, with an initial denaturing step of 8 min at 95° C.PCR products were then analyzed by agarose gel electrophoresis.

Results

This experiment provides a MSP assay to detect methylated andunmethylated DNA sequences of the androgen receptor gene. In total, 6male and 11 female health subjects were recruited. Of all male controlsubjects, only the unmethylated androgen receptor gene was detected inthese samples as expected (FIG. 1A). By contrast, both unmethylated andmethylated androgen receptor gene DNA sequences were observed in femalecontrol subjects (FIG. 1A). The detection rates of methylated andunmethylated androgen receptor genes in these female subjects were 100%and 82%, respectively. When DNA samples were omitted from MSP assay, nopositive signal was observed (FIG. 1A). Interestingly, positive signalsfor both methylated and unmethylated DNA sequences were observed in allmale sex-mismatched bone marrow transplantation recipients (100%),indicating cells from female donor exist in the blood circulation ofmale recipients.

These results demonstrate, for the first time that methylated genes onthe inactivated X chromosome from female individuals can be used as afemale-specific marker in chimerism research. This assay is alsoapplicable to the study of other types of post-transplantationchimerisms involving mixture of male and female cells or DNA. Examplesinclude cellular chimerism following solid organ transplantation (Starzlet al., Curr Opin Nephrol Hypertens 6:292-8 (1997)),post-transplantation plasma DNA chimerism (Lo et al., Lancet 351:1329-30(1998)) and urinary DNA chimerism (Zhang et al., Clin Chem 45: 1741-6(1995)). In addition, there is also much recent interest in the passageof cells and DNA from the mother into the fetus during pregnancy (Lo etal., Blood 88:4390-5. (1996); Maloney et al., J Clin Invest 104: 41-7(1999); Lo et al., Clin Chem 46:1301-9 (2000). The epigenetic markersdeveloped should also be of used in chimerism of maternal origin in maleoffsprings.

The current assay may be developed into a quantitative format, using forexample, real-time PCR technology (Lo et al., Cancer Res 59:3899-903(1999)). Such development would allow us to monitor the levels ofchimerism in a particular person. Clinically such an assay might have arole in the monitoring of graft acceptance in BMT. In the case ofurinary or plasma DNA chimerism, such an assay might also be used forthe monitoring of graft rejection.

EXAMPLE 2

Differential DNA Methylation Between Fetus and Mother as a Strategy forDetecting Fetal DNA in Maternal Plasma

The present experiment demonstrates that by using a differentiallymethylated region in the human IGF2-H19 locus as an epigenetic marker inmaternal plasma, detection of an allele that the fetus has inheritedfrom the mother is possible. These results greatly expand the prenataldiagnostic possibilities of fetal DNA in maternal plasma allowingdevelopment of a gender- and polymorphism-independent fetal-specificmarker in maternal plasma and new strategies for the prenatal diagnosisof imprinting disorders and certain chromosomal aneuploidies.

Materials and Methods

Subjects and Samples

Samples were collected from pregnant women with informed consent. Intotal, 21 and 18 women in the second trimester (17-21 weeks) and thirdtrimester (37-42 weeks) of pregnancy, respectively, were recruited forthis study. None of the recruited subjects had preeclampsia or pretermlabor in the current pregnancy. EDTA maternal blood and fetal amnioticfluid samples were collected from the second trimester cases asdescribed previously (Lo et al., Am J Hum Genet 62:768-775 (1998)). Forthe third trimester cases, we collected EDTA maternal blood samples at 2to 3 h before normal vaginal delivery. EDTA fetal cord blood sampleswere also collected immediately after delivery as described (Lo et al.,Clin Chem 46:1903-1906 (2000)). Plasma and buffy coat from all recruitedblood samples were harvested and stored at −20° C. as described (Lo etal., Am J Hum Genet 62:768-775 (1998)), except that plasma samples wererecentrifuged at 16,000 g. Amniotic fluid samples were stored at 4° C.

DNA Isolation

DNA was extracted from plasma and amniotic fluid samples using a QIAampBlood Kit (Qiagen). Typically, 800 μl of plasma or amniotic fluid wasused for DNA extraction per column. An elution volume of 50-110 μL wasused. DNA was extracted from the buffy coat using a Nucleon DNAExtraction Kit (Scotlabs) according to manufacturer's recommendations.

Genotyping of the DMR Polymorphic Region

The DMR in the human IGF2-H19 locus contains two 450-bp repeat and seven400-bp repeat units (Nakagawa et al., Proc Natl Acad Sci USA 98:591-596(2001)) (FIG. 2). An A/G SNP within the DMR (Nakagawa et al., supra) wasselected as a marker in our investigation (FIG. 2). Polymerase chainreaction (PCR) was used to amplify the SNP in both maternal and fetalDNA samples. Primers were designed using the sequence of the Homosapiens H19 gene (Genbank accession number AF125183). Typically, 2 to 5μl eluted DNA, purified from maternal buffy coat, cord buffy coat oramniotic fluid was added to a 25 μl PCR reaction containing 2.5 μl 10×TaqMan buffer A (PE Applied Biosystems), 3 mM MgCl₂, 6.26 pmol dNTPs, 5pmol primers (forward: 5′-ggACGGAATTGGTTGTAGTT-3′; reverse:5′-AGGCAATTGTCAGTTCAGTAA-3′) and 0.625 U AmpliTaq Gold DNA polymerase(PE Applied Biosystems) (95° C. for 8 min followed by 35 cycles of 95°C. for 1 min, 56° C. for 20 sec, 72° C. for 20 sec). For the forwardprimer, the nucleotides in upper case corresponded to positions 7927 to7944 of the H19 sequence (Genbank accession number AF125183). For thereverse primer, the nucleotides were complementary to positions 8309 to8329 of the H19 sequence. PCR products were then analysed by agarose gelelectrophoresis and DNA sequencing.

Bisulfite Conversion

Bisulfite modification of DNA samples was performed using a CpGenome DNAModification Kit (Intergen) as instructed by the manufacturer. Withbisulfite conversion, unmethylated cytosine residues would be convertedto uracil; while methylated cytosine residues would remain unchanged(Herman et al., Proc Natl Acad Sci USA 93:9821-9826 (1996)). Thesequence difference between methylated and unmethylated DNA followingbisulfite conversion could then be distinguished using different PCRprimers. In general, 1 μg of buffy coat DNA from maternal or cord blood,or 93 μl eluted DNA purified from maternal plasma or amniotic fluid wasused in a bisulfite conversion reaction. Bisulfite-treated DNA was theneluted in 25-50 μl 1μ Tris-EDTA.

Methylation-Specific PCR (MSP)

MSP assays were modified from the protocol as described (Herman et al.1996). Five μl bisulfite-treated DNA was added to a 50 μl PCR reactioncontaining 5 μl 10×TaqMan buffer A (PE Applied Biosystems), 2.5 mMMgCl₂, 10 pmol dNTPs, 20 pmol each of the corresponding MSP primers(FIG. 2) and 1.25 U AmpliTaq Gold DNA polymerase (PE AppliedBiosystems). The primers M-for and M-rev (FIG. 2) were designed for themethylated sequence, while the primers U-for and U-rev (FIG. 2) weredesigned for the unmethylated sequence. Reaction mixtures were thermalcycled (methylated allele: 95° C. for 45 sec, 55° C. for 20 sec, 72° C.for 20 sec; unmethylated allele: 95° C. for 45 sec, 49° C. for 20 sec,72° C. for 20 sec) for 50 (buffy coat and amniotic fluid DNA) or 56(plasma DNA) cycles, with an initial denaturing step of 8 min at 95° C.PCR products were then analyzed by agarose gel electrophoresis. Reactionproducts were purified using Microspin S-300 HR columns (AmershamPharmacia) for DNA sequencing or the primer extension assay.

DNA Sequencing

Purified PCR products were sequenced using an ABI Prism dRhodamineTerminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems)and the corresponding forward primers of the PCR products. Sequencingproducts were analysed using an ABI Prism 310 Genetic Analyser (PEApplied Biosystems).

Primer Extension Assay

Two μl of the purified MSP product was added to a 25 μl reactioncontaining 50 μM ddATP (2′,3′-dideoxyadenine triphosphate), 50 μM dGTP,50 μM dTTP, 0.2 pmol Cys-5-labeled primer(5′-GGGTTATTTGGGAATAGGATATTTA-3′), 4 U Thermo Sequenase (AmershamPharmacia) and 1.43 μl concentrated buffer. Reactions were thermalcycled for 40 cycles (95° C. for 30 sec, 51° C. for 20 sec, 72° C. for20 sec). The Cys-5-labeled primer was 25 nucleotides (nt) in length andthe polymorphic site was 2 nt away from the 3′-end of the primer. Forthe A allele, the incorporation of the ddATP at this polymorphic sitewould produce chain termination, thus resulting in an extension productof 27 nt (i.e., 25+2 nt). For the G allele, chain extension wouldcontinue until the next A residue which was 5 nt away from the 3′-end ofthe primer, thus resulting in an extension product of 30 nt (i.e., 25+5nt). Reaction products were electrophoresed using a 14% denaturingpolyacrylamide gel and analysed using an ALF Express Sequencer (AmershamPharmacia). Data were analysed by the AlleleLinks program (AmershamPharmacia).

Results

Genotyping of DMR

Thirty-nine pregnant women were recruited in this study. Maternalgenotype at the SNP within the DMR (FIG. 2) was determined by directsequencing of PCR products from the buffy coat DNA. The number ofpregnant women with each of the possible genotypes were 17 (GG, 43.6%),16 (AG, 41.0%) and 6 (AA, 15.4%).

Detection of Fetal DNA in Plasma from Women Heterozygous for a BiallelicPolymorphism

The 16 women who were heterozygous (i.e., AG) for the SNP were selectedfor further examination. As this is a biallelic polymorphism, thesewomen would not be considered informative at this polymorphic locus forthe detection of fetal DNA in maternal plasma, based on previouscriteria (Lo et al., Ann NY Acad Sci 731:204-213 (1994); Bianchi Am JHum Genet 62:763-764 (1998)). To demonstrate that differentialmethylation at this genomic region would allow us to overcome thislimitation, maternal DNA was bisulfite-treated and amplified by MSPusing the primers shown in FIG. 2. Similarly, fetal DNA isolated fromamniotic fluid (2nd trimester samples) or buffy coat of cord blood (3rdtrimester samples) was subjected to PCR and MSP to determine theimprinting status of the fetal alleles.

Amongst the 16 selected cases, the methylated (i.e.,paternally-inherited) alleles from four 3rd trimester and seven 2ndtrimester fetal samples were different from the methylated alleles ofthe respective mothers (FIG. 3 a,b; compare panels 1 and 2). To test ifthis differential methylation between fetus and mother would allow thefetal allele to be detected from maternal plasma, maternal plasma DNAfrom these cases was subjected to bisulfite conversion, followed by MSP.Interestingly, the paternally-inherited methylated fetal allele could bedetected in two 3rd trimester and four 2nd trimester maternal plasmasamples (FIG. 3 a,b; panels 3). To exclude the possibility that theseobservations were simply due to the existence of aberrantly methylatedmaternal DNA in maternal plasma, we collected a postnatal maternalplasma sample (˜3.5 years after delivery) from one of the positive casesfor further examination. We did not observe the additional methylatedallele in this postnatal sample (FIG. 3 a, panel 4), indicating that theadditional methylated allele in maternal sample during pregnancy was offetal origin. In addition, no positive signal was observed in the plasmaof non-informative cases (n=4, data not shown), thus furtherdemonstrating the specificity of this MSP assay. Taken together, thesedata indicate that the use of differential methylation between motherand fetus allows detecting fetal DNA in maternal plasma, even in caseswhich are not considered informative with existing criteria.

Detection of Fetal-Derived Maternally-Inherited DNA from Maternal Plasma

We then tested if the use of differential methylation between mother andfetus might allow us to detect an allele that the fetus has inheritedfrom the mother. This type of analysis has previously been thought to beimpossible (Lo et al., Ann NY Acad Sci 731:204-213 (1994); Bianchi, Am JHum Genet 62:763-764 (1998)). As the maternally-inherited allele wasunmethylated, the primers U-for and U-rev (FIG. 2) were used to amplifythe unmethylated allele following bisulfite conversion. Among the 16analyzed cases, three 3rd trimester and five 2nd trimester maternalsamples were informative. In these cases, the fetus possessed anunmethylated allele that was different from the unmethylated allele ofthe mother. These results implied that in these cases, the mother hadoriginally inherited the fetal allele from her father and then passed onto the fetus. Of these 8 informative cases, only a weak positive signalwas observed in one of the 3rd trimester samples on direct sequencing(FIG. 4 a, compared panel 1 and panel 2).

We reasoned that the weak signal in this single positive case and thelow detection rate of the unmethylated fetal allele from maternal plasmamight be due to the low sensitivity of the direct sequencing method. Toenhance the sensitivity of detection, we employed a more sensitiveprimer extension assay to detect the unmethylated fetal allele from theMSP reaction products. As the SNP was an A/G polymorphism, ddATP wasused as a reaction substrate in the primer extension assay. Extendedreaction products from the A and G alleles were 27 and 30 nt long,respectively. No fetal specific reaction product was present in thecorresponding maternal buffy coat samples (FIG. 4 b, c; maternal BC).Strikingly, fetal specific extension products were observed in two 3rdtrimester (FIG. 4 b, arrow) and one 2nd trimester (FIG. 4 c, arrow)maternal plasma samples, indicating the presence of unmethylated fetalDNA in maternal plasma. As controls, none of the tested non-informativecases was positive in this assay (n=5, data not shown). These resultsdemonstrated, for the first time, the feasibility of using epigeneticmarkers to detect a fetal-derived maternally-inherited DNA sequence frommaternal plasma.

Discussion

These results demonstrate that the use of epigenetic markers overcomesthe conventional limitations of detecting fetal DNA in maternal plasma.It is possible to detect a paternally-inherited fetal allele, which isgenetically indistinguishable from a maternal allele, from the mother'splasma, by the use of epigenetic differences between the mother andfetus. Likewise, it is possible to detect a maternally-inherited fetalallele from maternal plasma. This novel epigenetic approach willtherefore expand the repertoire of disorders wherein fetal DNA inmaternal plasma can be used.

Even with the use of relatively insensitive methods such as directsequencing and primer extension, the present results demonstrate that itis possible to detect differentially methylated fetal DNA sequences frommaternal plasma. There was a lower sensitivity in the detection of theunmethylated fetal DNA in maternal plasma (FIG. 4), as compared with theanalogous assay for the methylated allele (FIG. 3). Using more sensitivedetection systems, such as allele-specific PCR (Newton et al., NucleicAcids Res 17:2503-2516 (1989)) and real-time methylation-specific PCR(Lo et al., Cancer Res 59:3899-3903 (1999); Eads et al., Nucleic AcidsRes 28:E32 (2000)), might enhance the sensitivity of plasma-basedepigenetic analysis. The development of real-time methylation-specificPCR is particularly interesting as it opens up the possibility ofquantifying fetal-specific methylation in maternal plasma, as hasalready been achieved for the detection of tumor DNA in circulation(Kawakami et al., J Natl Cancer Inst 92:1805-1811 (2000)).

The possible introduction of fetal DNA in maternal plasma as a routineprenatal diagnostic tool has raised questions with regard to the need ofa generic marker for circulating fetal DNA (Lo et al., Am J Hum Genet62:768-775 (1998); Avent et al, Vox Sang 78:155-162 (2000)). Mostproposals for such a marker have thus far focused on the use of geneticpolymorphisms between the mother and fetus (Tang et al., Clin Chem45:2033-2035 (1999); Pertl et al., Hum Genet 106:45-49 (2000)). Thepresent demonstration of the feasibility of epigenetic markers for fetalDNA detection in maternal plasma opens up a new approach for thedevelopment of a gender-independent and polymorphism-independent fetalmarker in maternal plasma. One way wherein this can be achieved is toexploit the phenomenon of tissue-specific methylation (Grunau et al.,Hum Mol Genet 9:2651-2663 (2000)). As the trophoblast has been suggestedto be the predominant cell population for releasing fetal DNA intomaternal plasma, the elucidation of trophoblast-specific methylationpatterns allows the development of a generic epigenetic fetal marker inmaternal plasma. Biologically, the use of tissue-specific methylationmarkers may also allow one to directly address the question as to whatfetal cell types are responsible for releasing fetal DNA into maternalplasma.

The epigenetic analysis of maternal plasma has obvious applications todisorders associated with genomic imprinting, such as the Prader-Willisyndrome (Pfeifer, Lancet 356:1819-1820 (2000)). This strategy may alsohave diagnostic potential for disorders such as preeclampsia, whereinimprinted genes have been hypothesized to play a role (Graves, ReprodFertil Dev 10:23-29 (1998)).

The possible application of fetal DNA in maternal plasma for theprenatal detection of fetal chromosomal aneuploidies is an issue thathas been keenly discussed since the discovery of the phenomenon (Lo etal., Lancet 350:485-487 (1997); Bianchi, Am J Hum Genet 62:763-764(1998)). The finding of quantitative differences between the circulatingfetal DNA levels in aneuploid, compared with euploid pregnancies (Lo etal., Clin Chem 45:1747-1751 (1999); Zhong et al., Prenat Diagn20:795-798 (2000)) offers a method for estimating the risk of fetalchromosomal aneuploidies from maternal plasma. The recent discovery ofapoptotic fetal cells in maternal plasma (“plasma-derived cells”) (VanWijk et al., Clin Chem 46:729-731 (2000)) offers yet another approachfor aneuploidy detection from maternal plasma (Poon et al., Lancet356:1819-1820 (2000)). Interestingly, the present data open up yetanother potential approach for the detection of fetal chromosomalaneuploidies. This is based on the observation that aberrant DNAmethylation patterns may be associated with chromosomal aneuploidy(Kuromitsu et al., Mol Cell Biol 17:707-712 (1997); Yu et al., Proc NatlAcad Sci USA 94:6862-6867 (1997)). Hence it is possible to developepigenetic markers for detecting such aberrantly methylated fetal DNAsequences from maternal plasma. Such markers provide specificitycompared with a simple quantitation of fetal DNA in maternal plasma (Loet al., Clin Chem 45:1747-1751 (1999); Zhong et al., Prenat Diagn20:795-798 (2000)) and better suitability to large scale applicationcompared with methods based on “plasma-derived cells” (Poon et al.,Lancet 356:1819-1820 (2000)).

Fetal epigenetic markers may also be used in the analysis of fetal cellsisolated from the cellular fraction of maternal blood. This takesadvantage of recent data showing that methylation analysis could beperformed in an in situ manner (Nuovo et al., Proc Natl Acad Sci USA96:12754-12759 (1999)).

With the recent realization that fetomaternal trafficking is abidirectional process (Lo et al., Blood 88:4390-4395 (1996); Maloney etal., J Clin Invest 104:41-47 (1999)), epigenetic markers may also beused to investigate cellular and DNA transfer from the mother to thefetus. Such an approach might also have applications to theinvestigation of other types of chimerism, such as post-transplantationhemopoietic chimerism (Starzl et al., Curr Opin Nephrol Hypertens6:292-298 (1997)) and urinary DNA chimerism (Zhang et al., Clin Chem45:1741-1746 (1999)).

With our increased understanding of the human genome and the developmentof high throughput array-based technologies for methylation analysis(Yan et al., Clin Cancer Res 6:1432-1438 (2000)), we expect that thenumber of usable fetal epigenetic markers will rapidly increase over thenext few years. Such a development will provide a clinically relevantpanel of fetal epigenetic markers which can be used in a synergisticmanner with conventional genetic markers in maternal plasma.

1-39. (canceled)
 40. A kit for differentiating DNA species originatingfrom cells of different individuals, wherein the DNA species are in abiological sample obtained from one of the individuals, the kitcomprising: (i) a reagent for extracting the DNA species from thebiological sample; and (ii) a reagent for detecting a methylationdifference between the DNA species; wherein the detection of themethylation difference indicates DNA species from different individuals.41. The kit according to claim 40, wherein the reagent for detecting themethylation difference comprises sodium bisulfite.
 42. The kit accordingto claim 40, further comprising: (iii) a reagent for amplifying the DNAspecies.
 43. The kit according to claim 42, wherein the reagent foramplifying the DNA species is a reagent for performing a polymerasechain reaction to generate a PCR product.
 44. The kit according to claim42, wherein the reagent for amplifying the DNA species comprises areagent for performing a methylation-specific polymerase chain reaction.45. The kit according to claim 42, wherein the reagent for amplifyingthe DNA species comprises PCR primers distinguishing the sequencedifference between methylated and unmethylated DNA following bisulfiteconversion.
 46. The kit according to claim 42, wherein the reagent foramplifying the DNA species comprises a set of PCR primers designed for amethylated DNA sequence.
 47. The kit according to claim 42, wherein thereagent for amplifying the DNA species comprises a set of PCR primersdesigned for an unmethylated DNA sequence.
 48. The kit according toclaim 46, wherein the methylated DNA sequence is that of an androgenreceptor gene or IGF2-H 19 gene.
 49. The kit according to claim 47,wherein the unmethylated DNA sequence is that of an androgen receptorgene or IGF2-H₁₉ gene.
 50. The kit according to claim 43, furthercomprising: (iv) a reagent for sequencing the PCR product.
 51. The kitaccording to claim 43, further comprising: (iv) a reagent for purifyingthe PCR product.
 52. The kit according to claim 43, further comprising:(iv) a reagent for detecting the presence of the PCR product.
 53. Thekit according to claim 52, wherein the reagent for detecting thepresence of the PCR product is a gel.
 54. The kit according to claim 40,further comprising: (iii) a reagent for performing primer extension. 55.The kit according to claim 40, further comprising: (iii) a reagent forperforming real-time PCR.
 56. The kit according to claim 40, furthercomprising: (iii) a marker.
 57. The kit according to claim 40, whereinthe biological sample is a fluid or cellular sample or a mixturethereof.
 58. The kit according to claim 40, wherein the biologicalsample is plasma, serum, blood or amniotic fluid.
 59. The kit accordingto claim 58, further comprising: (iii) EDTA; wherein the EDTA is addedto the biological sample.
 60. The kit according to claim 40, wherein thecells of different individuals comprise cells of a transplantationrecipient and cells from an organ donor, wherein the DNA species are ina biological sample obtained from the transplantation recipient andwherein the detection of the methylation difference allows thedifferentiation of DNA species derived from the transplantationrecipient from those derived from the organ donor.
 61. A kit fordetecting a methylation difference between DNA species originating fromcells of a pregnant female and an unborn fetus, wherein the DNA speciesare in a biological sample obtained from either the pregnant female orunborn fetus, the kit comprising: (i) a reagent for extracting the DNAspecies from the biological sample; and (ii) a reagent for detecting amethylation difference between the DNA species; wherein the detection ofthe methylation difference is associated with a fetal or maternaldisorder.
 62. The kit according to claim 61, wherein the disorder ispreeclampsia, a chromosomal aneuploidy, trisomy 21 (Down syndrome),Prader Willi Syndrome or Angelman Syndrome.